IgA Fc binding protein

ABSTRACT

The invention provides IgA Fc binding protein polypeptides and DNA (RNA) encoding IgA Fc binding protein polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing IgA Fc binding protein polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This patent application claims benefit of U.S. Provisional PatentApplications 60/027,030, filed Sep. 24, 1996 and 60/040,656, filed Mar.10, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to polynucleotides and polypeptidesof the IgA Fc binding protein family, hereinafter referred to as "IgA Fcbinding protein".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

Several species of gram-positive pathogenic bacteria express cell wallproteins that bind to the Fc region of IgA in humans. Two of these fromStreptococcus have been studied in detail: protein Arp from the group AStreptococcus and protein Bac from the group B Streptococcus (seeLindahl, G., Akerstrom, B., Stenberg, L., Frithz, E and Heden, L. O.(1991) in Genetics and Molecular Biology of Streptococci, Lactococci andEnterococci (Dunni, G M et al., eds.) pp 155-159, American Soc.Microbiol. and also Jerlstrom, P. G., Chhatwal, G. S. and Timmis, K. N.(1991) Mol. Microbiol. 5: 843). Such proteins are valuable asimmunochemical tools as it is likely that they are used by the bacteriato evade the defense mechanisms of the infected host (see Stenberg L.,et al. (1994) J.Biol.Chem. 269(18): 13458).

Clearly, there is a need for factors, such as the compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown Streptococcus agalactiae IgA Fc binding protein protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as IgA Fc binding protein polypeptides of the invention byhomology between the amino acid sequence set out in Table 1 SEQ ID NO:2!and a known amino acid sequence or sequences of other proteins such asStreptococcus agalactiae IgA Fc binding protein protein.

It is a further object of the invention to provide polynucleotides thatencode IgA Fc binding protein polypeptides, particularly polynucleotidesthat encode the polypeptide herein designated IgA Fc binding protein.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding IgA Fc binding proteinpolypeptides comprising the sequence set out in Table 1 SEQ ID NO:1!, ora variant thereof.

In another particularly preferred embodiment of the invention there is aIgA Fc binding protein protein from Streptococcus pneumoniae comprisingthe amino acid sequence of Table 1 SEQ ID NO:2!, or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding IgA Fc binding protein, particularlyStreptococcus pneumoniae IgA Fc binding protein, including mRNAs, cDNAs,genomic DNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of IgA Fc binding protein and polypeptidesencoded thereby.

Another aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as IgA Fc binding protein aswell as biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of IgA Fc binding protein polypeptide encoded by naturallyoccurring alleles of the IgA Fc binding protein gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned IgA Fc binding protein polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing IgA Fcbinding protein expression, treating disease, for example, otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, assaying genetic variation,and administering a IgA Fc binding protein polypeptide or polynucleotideto an organism to raise an immunological response against a bacteria,especially a Streptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to IgA Fc binding protein polynucleotide sequences,particularly under stringent conditions.

In certain preferred embodiments of the invention there are providedantibodies against IgA Fc binding protein polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided IgA Fc binding protein agonists and antagonists, preferablybacteriostatic or bacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a IgA Fc binding protein polynucleotide or a IgA Fc bindingprotein polypeptide for administration to a cell or to a multicellularorganism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffn, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et aL, NCBI NLMNIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,ie., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism.is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to IgA Fc binding protein polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a IgA Fcbinding protein of Streptococcus pneumoniae, which is related by aminoacid sequence homology to Streptococcus agalactiae IgA Fc bindingprotein polypeptide. The invention relates especially to IgA Fc bindingprotein having the nucleotide and amino acid sequences set out in Table1 SEQ ID NO: 1! and Table 1 SEQ ID NO: 2! respectively, and to the IgAFc binding protein nucleotide sequences of the DNA in the depositedstrain and amino acid sequences encoded thereby.

                                      TABLE 1    __________________________________________________________________________    IgA Fc binding protein Polynucleotide and Polypeptide Sequences    __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae IgA Fc binding protein    polynucleotide sequence  SEQ ID NO:1!    5'-      1             CTGGGAGACA TTAAGCGAAC GTATTTGTAT AAATTAGATG AATCAACCCA            51             AAAAGCCCAA CTACAGGAAC TGGTCACAGA AAGTCAATCA AAACTAGATG           101             AAGCTTTTTC TAAATTTAAA AATGGCTTAT CTTCTTCGTC AAGTTCAGGA           151             TCCTCCACTA AACCAGAAAC TCCGCAGCCG GAAAAACCAG AGCATCAAAA           201             ACCAACAACT CCAGCTCCGG ATACCAAACC AAGCCCTCAA CCAGAAGGCA           251             AGAAACCAAG CGTACCAGAC ATTAATCAGG AAAAAGAAAA AGCTAAGCTT           301             GCTGTAGCAA CCTACATGAG CAAGATTTTA GATGATATAC AAAAACATCA           351             TCTGCAGAAA GAAAAACATC GTCAGATTGT TGCTCTTATT AAGGAGCTTG           401             ATGAGCTTAA AAAGCAAGCT CTTTCTGAAA TTGATAATGT AAATACCAAA           451             GTAGAAATTG AAAATACAGT CCACAAGATA TTTGCAGACA TGGATGCAGT           501             TGTGACTAAA TTCAAAAAAG GCTTAACTCA GGACACACCA AAAGAACCAG           551             GTAACAAAAA ACCATTTGCT CCAAAACCAG GTATGCAACC AAGTCCTCAA           601             CCAGAGGTTA AACCGCAGCT AGAAAAACCA AAACCAGAGG TTAAACCGCA           651             ACCAGAAAAA CCAAAACCAG AGGTTAAACC GCAACCAGAA AAACCAAAAC           701             CAGAGGTTAA ACCGCAGCCG GAAAAACCAA AACCAGAGGT TAAACCGCAA           751             CCAGAAAAAC CAAAACCAGA GGTTAAACCG CAGCCGGAAA AACCAAAACC           801             AGAGGTTAAA CCGCAGCTGG AAAAACCAAA ACCAGATAAT AGCAAGCCAC           851             AAGCAGATGA TAAGAAGCCA TCAACTACAA ATAATTTAAG CAAGGACAAG           901             CAACCTTCTA ACCAAGCTTC AACAAACGAA AAAGCAACAA ATAAACCGAA           951             GAAGTCATTG CCATCAACTG GATCTATTTC AAATCTAGCA CTTGAAATTG          1001             CAGGTCTTCT TACCTTGGCG GGGGCAACCA TTCTTGCTAA GAAAAGAATG          1051             AAATAG-3'    (B) IgA Fc binding protein polypeptide sequence deduced from    the polynucleotide sequence in this table  SEQ ID NO:2!    NH.sub.2 -             1             LGDIKRTYLY KLDESTQKAQ LQELVTESQS KLDEAFSKFK NGLSSSSSSG            51             SSTKPETPQP EKPEHQKPTT PAPDTKPSPQ PEGKKPSVPD INQEKEKAKL           101             AVATYMSKIL DDIQKHHLQK EKHRQIVALI KELDELKKQA LSEIDNVNTK           151             VEIENTVHKI FADMDAVVTK FKKGLTQDTP KEPGNKKPFA PKPGMQPSPQ           201             PEVKPQLEKP KPEVKPQPEK PKPEVKPQPE KPKPEVKPQP EKPKPEVKPQ           251             PEKPKPEVKP QPEKPKPEVK PQLEKPKPDN SKPQADDKKP STTNNLSKDK           301             QPSNQASTNE KATNKPKKSL PSTGSISNLA LEIAGLLTLA GATILAKKRM           351             K-COOH    (C) Polynucleotide sequence embodiments  SEQ ID NO:1!    X-(R.sub.1).sub.n -             1             CTGGGAGACA TTAAGCGAAC GTATTTGTAT AAATTAGATG AATCAACCCA            51             AAAAGCCCAA CTACAGGAAC TGGTCACAGA AAGTCAATCA AAACTAGATG           101             AAGCTTTTTC TAAATTTAAA AATGGCTTAT CTTCTTCGTC AAGTTCAGGA           151             TCCTCCACTA AACCAGAAAC TCCGCAGCCG GAAAAACCAG AGCATCAAAA           201             ACCAACAACT CCAGCTCCGG ATACCAAACC AAGCCCTCAA CCAGAAGGCA           251             AGAAACCAAG CGTACCAGAC ATTAATCAGG AAAAAGAAAA AGCTAAGCTT           301             GCTGTAGCAA CCTACATGAG CAAGATTTTA GATGATATAC AAAAACATCA           351             TCTGCAGAAA GAAAAACATC GTCAGATTGT TGCTCTTATT AGGAGCTTG           401             ATGAGCTTAA AAAGCAAGCT CTTTCTGAAA TTGATAATGT AAATACCAAA           451             GTAGAAATTG AAAATACAGT CCACAAGATA TTTGCAGACA TGGATGCAGT           501             TGTGACTAAA TTCAAAAAAG GCTTAACTCA GGACACACCA AAAGAACCAG           551             GTAACAAAAA ACCATTTGCT CCAAAACCAG GTATGCAACC AAGTCCTCAA           601             CCAGAGGTTA AACCGCAGCT AGAAAAACCA AAACCAGAGG TTAAACCGCA           651             ACCAGAAAAA CCAAAACCAG AGGTTAAACC GCAACCAGAA AAACCAAAAC           701             CAGAGGTTAA ACCGCAGCCG GAAAAACCAA AACCAGAGGT TAAACCGCAA           751             CCAGAAAAAC CAAAACCAGA GGTTAAACCG CAGCCGGAAA AACCAAAACC           801             AGAGGTTAAA CCGCAGCTGG AAAAACCAAA ACCAGATAAT AGCAAGCCAC           851             AAGCAGATGA TAAGAAGCCA TCAACTACAA ATAATTTAAG CAAGGACAAG           901             CAACCTTCTA ACCAAGCTTC AACAAACGAA AAAGCAACAA ATAAACCGAA           951             GAAGTCATTG CCATCAACTG GATCTATTTC AAATCTAGCA CTTGAAATTG          1001             CAGGTCTTCT TACCTTGGCG GGGGCAACCA TTCTTGCTAA GAAAAGAATG          1051             AAATAG-(R.sub.2).sub.n -Y    (D) Polypeptide sequence embodiments  SEQ ID NO:2!.    X-(R.sub.2).sub.n -             1             LGDIKRTYLY KLDESTQKAQ LQELVTESQS KLDEAFSKFK NGLSSSSSSG            51             SSTKPETPQP EKPEHQKPTT PAPDTKPSPQ PEGKKPSVPD INQEKEKAKL           101             AVATYMSKIL DDIQKHHLQK EKHRQIVALI KELDELKKQA LSEIDNVNTK           151             VEIENTVHKI FADMDAVVTK FKKGLTQDTP KEPGNKKPFA PKPGMQPSPQ           201             PEVKPQLEKP KPEVKPQPEK PKPEVKPQPE KPKPEVKPQP EKPKPEVKPQ           251             PEKPKPEVKP QPEKPKPEVK PQLEKPKPDN SKPQADDKKP STTNNLSKDK           301             QPSNQASTNE KATNKPKKSL PSTGSISNLA LEIAGLLTLA GATILAKKRM           351             K-(R.sub.2).sub.n -Y    __________________________________________________________________________

Deposfted materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus peumnoniae 0100993 on deposit.

On Apr. 17, 1996 a Streptococcus peumnoniae 0100993 DNA library in E.coli was similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas "the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length IgA Fc binding proteingene. The sequence of the polynucleotides contained in the depositedstrain, as well as the amino acid sequence of the polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1 SEQID NO:2! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofIgA Fc binding protein, and also those which have at least 70% identityto the polypeptide of Table 1 SEQ ID NO:2! or the relevant portion,preferably at least 80% identity to the polypeptide of Table 1 SEQ IDNO:2!, and more preferably at least 90% similarity (more preferably atleast 90% identity) to the polypeptide of Table 1 SEQ ID NO:2! and stillmore preferably at least 95% similarity (still more preferably at least95% identity) to the polypeptide of Table 1 SEQ ID NO:2! and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) wherein, at the amino terminus, X is hydrogen, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is any amino acidresidue, and n is an integer between 1 and 1000. Any stretch of aminoacid residues denoted by either R group, where R is greater than 1, maybe either a heteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with IgA Fc binding proteinpolypeptides fragments may be "free-standing," or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of the amino acid sequence of Table 1 SEQ ID NO:2!, or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Streptococcus pneumoniae, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of IgA Fc binding protein, includingthose with a similar activity or an improved activity, or with adecreased undesirable activity. Also included are those fragments thatare antigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides thatencode the IgA Fc binding protein polypeptide having the deduced aminoacid sequence of Table 1 SEQ ID NO:2! and polynucleotides closelyrelated thereto and variants thereof.

Using the information provided herein, such as the polynucleotidesequence set out in Table 1 SEQ ID NO:1!, a polynucleotide of theinvention encoding IgA Fc binding protein polypeptide may be obtainedusing standard cloning and screening methods, such as those for cloningand sequencing chromosomal DNA fragments from bacteria usingStreptococcus pneumoniae 0100993 cells as starting material, followed byobtaining a full length clone. For example, to obtain a polynucleotidesequence of the invention, such as the sequence given in Table 1 SEQ IDNO:1!, typically a library of clones of chromosomal DNA of Streptococcuspneumoniae 0100993 in E. coli or some other suitable host is probed witha radiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York (1989). (see in particular ScreeningBy Hybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 SEQ ID NO:1! was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 SEQ ID NO:1! contains an openreading frame encoding a protein having about or exactly the number ofamino acid residues set forth in Table 1 SEQ ID NO:2! with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The open reading framemay be correlated with the polypeptide of Table 1 SEQ ID NO: 2! by usinga codon chart well known in the art or any algorithm for detecting openreading frames, such as the known methods of Framesearch (WisconsinPackage of the Genetics Computer Group (GCG) software), TBLASTN (Blastsoftware from NCBI), and FASTA (TFASTX of Pearson & Lipman; PNAS 85:2444 (1988).

IgA Fc binding protein of the invention is structurally related to otherproteins of the IgA Fc binding protein family, as shown by the resultsof sequencing the DNA encoding IgA Fc binding protein of the depositedstrain. The protein exhibits greatest homology to Streptococcusagalactiae IgA Fc binding protein protein among known proteins. IgA Fcbinding protein of Table 1 SEQ ID NO:2! has about 50% identity over itsentire length and about 60 % similarity over its entire length with theamino acid sequence of Streptococcus agalactiae IgA Fc binding proteinpolypeptide (over amino acid 722-822 of S. agalactiae IgA Fc bindingprotein).

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 SEQ ID NO:1!. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is the polynucleotide comprisingnucleotide set forth in SEQ ID NO:1 of Table 1 which encodes the IgA Fcbinding protein polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C) wherein, at the 5' end of the molecule, X is hydrogen, andat the 3' end of the molecule, Y is hydrogen or a metal, R₁ and R₂ isany nucleic acid residue, and n is an integer between 1 and 1000. Anystretch of nucleic acid residues denoted by either R group, where R isgreater than 1, may be either a heteropolymer or a homopolymer,preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae IgA Fcbinding protein having the amino acid sequence set out in Table 1 SEQ IDNO:2!. The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or an insertion sequence orediting) together with additional regions, that also may contain codingand/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 SEQ ID NO:2!. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingIgA Fc binding protein variants, that have the amino acid sequence ofIgA Fc binding. protein polypeptide of Table 1 SEQ ID NO:2! in whichseveral, a few, 5 to 10, to 5, to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of IgA Fc binding protein.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding IgA Fc binding protein polypeptide having the amino acidsequence set out in Table 1 SEQ ID NO:2!, and polynucleotides that arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical over its entire length to a polynucleotide encoding IgA Fcbinding protein polypeptide of the deposited strain and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical over their entire length to the same are particularlypreferred, and among these particularly preferred polynucleotides, thosewith at least 95% are especially preferred. Furthermore, those with atleast 97% are highly preferred among those with at least 95%, and amongthese those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 SEQ ID NO:1!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said DNA sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding IgA Fcbinding protein and to isolate cDNA and genomic clones of other genesthat have a high sequence similarity to the IgA Fc binding protein gene.Such probes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less.

For example, the coding region of the IgA Fc binding protein gene may beisolated by screening using the DNA sequence provided in SEQ ID NO: 1 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the IgA Fc binding proteinpolynucleotides of the invention for use as diagnostic reagents.Detection of IgA Fc binding protein in a eukaryote, particularly amammal, and especially a human, will provide a diagnostic method fordiagnosis of a disease. Eukaryotes (herein also "individual(s)"),particularly mammals, and especially humans, particularly those infectedor suspected to be infected with an organism comprising the IgA Fcbinding protein gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled IgA Fc binding protein polynucleotidesequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in the electrophoretic mobility of the DNA fragments ingels, with or without denaturing agents, or by direct DNA sequencing.See, e.g., Myers et al., Science, 230: 1242 (1985). Sequence changes atspecific locations also may be revealed by nuclease protection assays,such as RNase and S1 protection or a chemical cleavage method. See, eg.,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding IgA Fc binding protein can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2.

                  TABLE 2    ______________________________________    Primers for amphlication of IgA Fc binding protein polynucleotides    SEQ ID NO   PRIMER SEQUENCE    ______________________________________    3           5'-ATTAAGCGAA                             CGTATTTGTA-3'    4           5'-GGAATTTAGT                             CACAACTGCA-3'    ______________________________________

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying IgA Fc binding protein DNAisolated from a sample derived from an individual. The primers may beused to amplify the gene isolated from an infected individual such thatthe gene may then be subject to various techniques for elucidation ofthe DNA sequence. In this way, mutations in the DNA sequence may bedetected and used to diagnose infection and to serotype and/or classifythe infectious agent.

The invention furter provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having the sequence of Table 1 SEQ ID NO: 1!. Increasedor decreased expression of IgA Fc binding protein polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting and other hybridizationmethods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of IgA Fc binding protein protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a IgA Fc binding protein protein, in a sample derived from ahost are well-known to those of skill in the art. Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-IgA Fc binding protein or from naivelibraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J.et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by chain shuffling (Clackson, T. et al.,(1991) Nature 352, 624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against IgA Fc binding protein-polypeptide may be employed to treat infections, particularly bacterialinfections and especially otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarily determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and agonists--assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of IgA Fcbinding protein polypeptides or polynucleotides, particularly thosecompounds that are bacteristatic and/or bacteriocidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagoists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising IgA Fc binding proteinpolypeptide and a labeled substrate or ligand of such polypeptide isincubated in the absence or the presence of a candidate molecule thatmay be a IgA Fc binding protein agonist or antagonist. The ability ofthe candidate molecule to agonize or antagonize the IgA Fc bindingprotein polypeptide is reflected in decreased binding of the labeledligand or decreased production of product from such substrate. Moleculesthat bind gratuitously, i.e., without inducing the effects of IgA Fcbinding protein polypeptide are most likely to be good antagonists.Molecules that bind well and increase the rate of product productionfrom substrate are agonists. Detection of the rate or level ofproduction of product from substrate may be enhanced by using a reportersystem. Reporter systems that may be useful in this regard include butare not limited to calorimetric labeled substrate converted intoproduct, a reporter gene that is responsive to changes in IgA Fc bindingprotein polynucleotide or polypeptide activity, and binding assays knownin the art.

Another example of an assay for IgA Fc binding protein antagonists is acompetitive assay that combines IgA Fc binding protein and a potentialantagonist with IgA Fc binding protein-binding molecules, recombinantIgA Fc binding protein binding molecules, natural substrates or ligands,or substrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. IgA Fc binding protein can be labeled,such as by radioactivity or a colorimetric compound, such that thenumber of IgA Fc binding protein molecules bound to a binding moleculeor converted to product can be determined accurately to assess theeffectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing IgA Fc binding protein-induced activities, thereby preventingthe action of IgA Fc binding protein by excluding IgA Fc binding proteinfrom binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56.: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANHISENSEINHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of IgA Fc binding protein.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock IgA Fc binding protein protein-mediated mammalian cell invasionby, for example, initiating phosphorylation of mammalian tyrosinekinases (Rosenshine et al., Infect. Immun. 60:2211 (1992); to blockbacterial adhesion between mammalian extracellular matrix proteins andbacterial IgA Fc binding protein proteins that mediate tissue damageand; to block the normal progression of pathogenesis in infectionsinitiated other than by the implantation of in-dwelling devices or byother surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofIgA Fc binding protein) found using screens provided by the invention,particularly broad-spectrum antibiotics, should be useful in thetreatment of H. pylori infection. Such treatment should decrease theadvent of H. pylori-induced cancers, such as gastrointestinal carcinoma.Such treatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with IgA Fc binding protein, or afragment or variant thereof, adequate to produce antibody and/ or T cellimmune response to protect said individual from infection, particularlybacterial infection and most particularly Streptococcus pneumoniaeinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of IgA Fc binding protein, or a fragment or a variantthereof, for expressing IgA Fc binding protein, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/ or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

Provided by the invention are methods using the described polynucleotideor particular fragments thereof that have been shown to encodenon-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunisation experiments in animalmodels of infection with S. pneumoniae. The methods will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response in animals. It is forseen that thisapproach will allow for the subsequent preparation of monoclonalantibodies of particular value from the requisite organ of the animalsuccessfully resisting or clearing infection for the development ofprophylactic agents or therapeutic treatments of S. pneumoniae infectionin mammals, particularly humans.

A furter aspect of the invention relates to an immunological compositionwhich, when introduced into an individual capable or having inducedwithin it an immunological response, induces an immunological responsein such individual to a IgA Fc binding protein or protein codedtherefrom, wherein the composition comprises a recombinant IgA Fcbinding protein or protein coded therefrom comprising DNA which codesfor and expresses an antigen of said IgA Fc binding protein or proteincoded therefrom. The immunological response may be used therapeuticallyor prophylactically and may take the form of antibody immunity orcellular immunity such as that arising from CTL or CD4+T cells.

A IgA Fc binding protein polypeptide or a fragment thereof may be fusedwith co-protein which may not by itself produce antibodies, but iscapable of stabilizing the first protein and producing a fused proteinwhich will have immunogenic and protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Hemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain IgA Fcbinding protein protein, it is to be understood that this coversfragments of the naturally occurring protein and similar proteins withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. The sequencing data from two or more clonescontaining overlapping Streptococcus pneumoniae DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapiE that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1056 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #AAAAGCCCAA    60AGCGAAC GTATTTGTAT AAATTAGATG AATCAACCCA    #TAAATTTAAA   120TCACAGA AAGTCAATCA AAACTAGATG AAGCTTTTTC    #TCCGCAGCCG   180CTTCGTC AAGTTCAGGA TCCTCCACTA AACCAGAAAC    #AAGCCCTCAA   240ATCAAAA ACCAACAACT CCAGCTCCGG ATACCAAACC    #AGCTAAGCTT   300AACCAAG CGTACCAGAC ATTAATCAGG AAAAAGAAAA    #TCTGCAGAAA   360ACATGAG CAAGATTTTA GATGATATAC AAAAACATCA    #AAAGCAAGCT   420AGATTGT TGCTCTTATT AAGGAGCTTG ATGAGCTTAA    #CCACAAGATA   480ATAATGT AAATACCAAA GTAGAAATTG AAAATACAGT    #GGACACACCA   540ATGCAGT TGTGACTAAA TTCAAAAAAG GCTTAACTCA    #AAGTCCTCAA   600ACAAAAA ACCATTTGCT CCAAAACCAG GTATGCAACC    #ACCAGAAAAA   660CGCAGCT AGAAAAACCA AAACCAGAGG TTAAACCGCA    #ACCGCAGCCG   720TTAAACC GCAACCAGAA AAACCAAAAC CAGAGGTTAA    #GGTTAAACCG   780CAGAGGT TAAACCGCAA CCAGAAAAAC CAAAACCAGA    #ACCAGATAAT   840CAAAACC AGAGGTTAAA CCGCAGCTGG AAAAACCAAA    #CAAGGACAAG   900CAGATGA TAAGAAGCCA TCAACTACAA ATAATTTAAG    #GAAGTCATTG   960AAGCTTC AACAAACGAA AAAGCAACAA ATAAACCGAA    #TACCTTGGCG  1020CTATTTC AAATCTAGCA CTTGAAATTG CAGGTCTTCT    #     1056         GCTAA GAAAAGAATG AAATAG    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 351 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #Lys Leu Asp Glu Ser Thrrg Thr Tyr Leu Tyr    #                 15    #Glu Ser Gln Ser Lys Leuln Glu Leu Val Thr    #             30    #Leu Ser Ser Ser Ser Serys Phe Lys Asn Gly    #         45    #Gln Pro Glu Lys Pro Gluys Pro Glu Thr Pro    #     60    #Thr Lys Pro Ser Pro Glnhr Pro Ala Pro Asp    # 80    #Ile Asn Gln Glu Lys Gluro Ser Val Pro Asp    #                 95    #Ser Lys Ile Leu Asp Aspal Ala Thr Tyr Met    #            110    #His Arg Gln Ile Val Alaeu Gln Lys Glu Lys    #        125    #Gln Ala Leu Ser Glu Ilesp Glu Leu Lys Lys    #    140    #Asn Thr Val His Lys Ileys Val Glu Ile Glu    #160    #Phe Lys Lys Gly Leu Thrla Val Val Thr Lys    #                175    #Lys Pro Phe Ala Pro Lyslu Pro Gly Asn Lys    #            190    #Val Lys Pro Gln Leu Gluer Pro Gln Pro Glu    #        205    #Glu Lys Pro Lys Pro Glual Lys Pro Gln Pro    #    220    #Glu Val Lys Pro Gln Prolu Lys Pro Lys Pro    #240    #Pro Glu Lys Pro Lys Prolu Val Lys Pro Gln    #                255    #Pro Glu Val Lys Pro Glnro Glu Lys Pro Lys    #            270    #Pro Gln Ala Asp Asp Lysro Asp Asn Ser Lys    #        285    #Asp Lys Gln Pro Ser Asnsn Asn Leu Ser Lys    #    300    #Lys Pro Lys Lys Ser Leulu Lys Ala Thr Asn    #320    #Leu Glu Ile Ala Gly Leule Ser Asn Leu Ala    #                335    #Lys Lys Arg Met Lysly Ala Thr Ile Leu Ala    #            350    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    # 20               TTGTA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    # 20               CTGCA    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:2.
 2. Anisolated nucleic acid segment comprising a nucleotide sequence which isfully complementary to the polynucleotide segment of claim
 1. 3. Anisolated vector comprising the polynucleotide segment of claim
 1. 4. Anisolated vector comprising the nucleic acid segment of claim
 2. 5. Anisolated host cell comprising the vector of claim
 3. 6. An isolated hostcell comprising the vector of claim
 4. 7. A process for producing an IgAFc binding polypeptide encoded by said polynucleotide segment comprisingculturing the host cell of claim 5 under conditions sufficient for theproduction of said polypeptide.
 8. An isolated polynucleotide segmentcomprising a nucleotide sequence which is identical to the referencesequence of SEQ ID NO:1, except that the nucleotide sequence includes upto five nucleotide substitutions, insertions or deletions for every 100nucleotides of the reference sequence of SEQ ID NO:1.
 9. An isolatedpolynucleotide segment comprising a nucleotide sequence which isidentical to the reference sequence of SEQ ID NO:1, except that thenucleotide sequence includes up to ten nucleotide substitutions,insertions or deletions for every 100 nucleotides of the referencesequence of SEQ ID NO:1.
 10. An isolated polynucleotide segmentcomprising a nucleotide sequence which is identical to the referencesequence of SEQ ID NO:1, except that the nucleotide sequence includes upto twenty nucleotide substitutions, insertions or deletions for every100 nucleotides of the reference sequence of SEQ ID NO:1.
 11. Anisolated polynucleotide segment comprising a nucleotide sequence whichis identical to the reference sequence of SEQ ID NO:1, except that thenucleotide sequence includes up to thirty nucleotide substitutions,insertions or deletions for every 100 nucleotides of the referencesequence of SEQ ID NO:1.
 12. An isolated polynucleotide segmentcomprising a nucleotide sequence encoding the same mature polypeptideexpressed by the gene corresponding to the sequence of SEQ ID NO:1contained in Streptococcus pneumoniae 0100993 contained in NCIMB Depositnumber
 40794. 13. An isolated polynucleotide segment comprising anucleotide sequence from position 1 to 1053 inclusive of thepolynucleotide sequence set forth in SEQ ID NO:1.
 14. An isolatednucleic acid segment comprising a nucleotide sequence which is fullycomplementary to the polynucleotide segment of claim 8, 11, 12 or 13.15. An isolated vector comprising the polynucleotide segment of claims8, 9, 11, 12 or
 13. 16. An isolated vector comprising the nucleic acidsegment of claim
 14. 17. An isolated host cell comprising the vector ofclaim
 15. 18. A process for producing an IgA Fc binding polypeptideencoded by said polynucleotide segment comprising culturing the hostcell of claim 17 under conditions sufficient for the production of saidpolypeptide.